Many biological techniques such as are employed in biotechnology, microbiology, clinical diagnostics and treatment, in vitro fertilization, hematology and pathology, require such processes as identification, separation, culturing, or manipulation of a target entity such as a type of cell or microbe within a fluid medium such as blood, other bodily fluids, culture fluids or samples from the environment. It is often desirable to retain viability of the target entity or to culture the target entity.
Identification techniques typically involve labelling the target entity with a reagent which can be detected according to a characteristic property. Entities which can be viewed optically such as cells or certain microbes, may be identified using fluorescent MAb's or staining reagents specific to certain classes of cells or microbes. When such identification is done manually or mechanically, as by microscopy, multiple operations involving incubations and washing steps to remove excess labelling reagent are often performed. For example, in the usual method used to identify a subset of T-lymphocytes, such as T-Helper Cells or CD4-positive cells, a mixture of peripheral blood lymphocytes is incubated with a fluorescent MAb directed to CD4-positive cells. The MAb is then given sufficient incubation time to react with the CD4-positive cells. The CD4-positive cells are then washed using multiple centrifugations and can then readily be identified by fluorescent microscopy.
In the practice of manual fluorescent labelling methods employing a fluorescent microscope, direct labeling with MAb's is often impractical due to the expense of obtaining a cell-specific fluorescent Mab and because of reduced signal availability. Thus the technique of indirect analysis is common. During indirect analysis, the target entities are first labeled with a specific non-fluorescent MAb. Excess Mab is washed away. Then, a fluorescent-labeled second reagent such as fluorescent-labeled goat anti-mouse antibody is added to the medium. The medium is allowed to incubate to allow the labelled second reagent to bind with the non-fluorescent MAb and then excess reagent is removed. The target entities may then be identified due to the attachment of the fluorescent secondary reagent to the non-fluorescent biospecific MAb. Such methods are time-consuming, costly, and require considerable quantities of reagents. Moreover, as the number of operations employed in such identification processes increases, a greater number of target entities are lost or killed. Accurate microbial analyses employing such methodologies are difficult to achieve because of the small numbers of target entities involved as well as the difficulty of washing away unbound labeling agents. Other methodologies such as flow cytometry (fluorescent activated cell sorting) or field flow fractionation can be used for such analysis and in some instances require fewer manipulations. These other methods, however, require expensive equipment, highly trained personnel and typically can only analyze or separate one sample at a time.
Manipulation of target entities required by other biological techniques may also involve such processes as insertion of genetic material, organelles, subcellular components, viruses, or other foreign materials or bodies into the target entities. Inserted materials can be labeled prior to insertion so that effects and movements of these materials can be studied during incubation of the medium. In techniques such as transfection, or in vitro fertilization mechanical probes or arms are often used to hold the target entities. Such mechanical holding methods tend to obscure or damage the target entities.
It would be desirable in such biotechnical procedures as have been discussed to provide devices and methods for precise non-destructive immobilization and manipulation of specific target entities in an inexpensive and rapid manner.
Magnetic colloids having particles coated with biospecific compounds which attach to target entities are known to be useful in certain biospecific separation techniques. Reaction rates between such colloidal particles and target entities can be relatively rapid due to fast kinetic activity of the particles and sufficiently large areas of exposed reactant coatings. Magnetic particles in the range of 0.7 to 1.5 microns have been described in U.S. Pat. Nos. 3,790,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents, having reasonably good suspension characteristics when mildly agitated. Magnetic particle suspensions presently in commercial use tend to flocculate in time and must be resuspended by agitation prior to use. Such agitation adds another step to any process employing such reagents.
Small magnetic particles, such as those mentioned above, generally fall into two broad categories: particles that are permanently magnetized; and particles that become magnetic when subjected to a magnetic field. The latter particles are referred to herein as magnetically-responsive particles. Materials displaying magnetically-responsive behavior are sometimes described as superparamagnetic. However, certain ferromagnetic materials such as magnetic iron oxide crystals, behave in a magnetically-responsive manner when the crystals are less than about 30 nm in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. The properties of such particles are described in P. Robinson et al., Biotech Bioeng. XV:603-06 (1973).
Magnetically-responsive colloidal magnetite is disclosed in U.S. Pat. No. 4,795,698 to Owen et al., which relates to polymer-coated, sub-micron size magnetite particles that behave as true colloids. Several devices are known which are used to separate magnetic particles from colloidal suspensions. Examples of such devices are magnetic separators such as the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass.; the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y.; and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass. A similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass. is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each test tube fitting into a recess between two of the bar magnets.
The above-described magnetic separators have the disadvantage that the magnetic particles and other impurities tend to form several layers on the inner surface of the sample container where they become entrapped and are difficult to remove even with vigorous washing. These separators are also not capable of establishing monolayers of target entities for microscopic analysis or manipulation.
Separation of magnetically-responsive particles within colloidal suspensions requires high gradient field separation techniques such as are described in R. R. Oder, IEEE Trans. Magnetics, 12:428-35 (1976); C. Owen and P. Liberti, Cell Separation: Methods and Selected Applications, Vol. 5, Pretlow and Pretlow eds., Academic Press, NY, (1986). Gradient fields normally used to filter such materials generate relatively large magnetic forces. Another useful technique for performing magnetic separation of colloidal magnetic particles from a test medium by the addition of agglomerating agents is disclosed in and commonly-owned U.S. Pat. No. 5,108,933 issued Apr. 28, 1992.
A commercially available high gradient magnetic separator, the MACS device made by Miltenyi Biotec GmbH, Gladback, West Germany, employs a column filled with a non-rigid steel wool matrix in cooperation with a permanent magnet. In operation, the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic components of the test medium pass through the column. It has been found that the steel wool matrix of such prior art high-gradient magnetic separation (HGMS) devices often causes non-specific entrapment of biological entities other than the target entities. The entrapped non-magnetic components cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance. Moreover, the sizes of the columns in many of the prior art HGMS devices require substantial volumes of test media, which poses an impediment to their use in performing various useful laboratory- scale separations. In addition, the steel wool matrix may damage sensitive cell types.
Although HGMS affords certain advantages in performing medical or biological analyses based on biospecific affinity reactions involving colloidal magnetic particles, the systems developed to date are not particularly suited for immobilization and micromanipulation. For example, collection of microscopic entities upon an irregular structure such as steel wool is not conducive to microscopic observation wherein it is desirable to maintain the subject of interest in the focal plane of a microscope. Furthermore, the convoluted surface of the steel wool would obscure observation of the collected entities. Accordingly, it would be desirable to provide an HGMS apparatus for immobilization and micromanipulation of target entities which is of relatively simple construction and operation and yet maximizes magnetic field gradients, so as to be of practical utility in conducting various laboratory-scale separations and micromanipulations.